Project Summary/Abstract The ability of neurons to send and receive signals underlies the most essential functions of the brain, from regulating breathing to complex thought. In order for the brain to function properly, the formation and function of new synapses must be tightly regulated. One mechanism that can contribute to this regulation is local protein synthesis, which allows for finely tuned and spatially precise responses to changes in the neuron?s environment. It has recently been discovered that the local synthesis of synaptosomal-associated protein 25 (SNAP25) at the presynaptic site is required for the formation and function of presynaptic terminals. However, the mechanisms controlling SNAP25 mRNA, localization, stability, and translation are largely unknown. SNAP25 has previously been implicated in several neuropsychiatric disorders, in particular attention deficit/hyperactivity disorder (ADHD). Reductions in SNAP25 have also been associated with schizophrenia and epilepsy. In vitro, blocking the translation of Snap25 specifically in axons prevents the clustering of presynaptic proteins at the presynaptic terminal and slows vesicular release, indicating that the post- transcriptional regulation of Snap25 is crucial for synapse development. The RNA-binding proteins Pumilio (Pum) 1 and 2 regulate Snap25 mRNA by binding to its 3?UTR. Traditionally known as translational repressors, Pum1 and Pum2 have also recently been identified as negative regulators of the axonal transcriptome. Therefore, I hypothesize that de-repression and presynaptic localization of Snap25 by developmental downregulation of Pumilio proteins is required for proper control of presynaptic terminal formation and function, and that disruption of these regulatory mechanisms will lead to aberrant presynaptic terminal formation and synaptic release. I will address this hypothesis through the following Specific Aims: Aim 1 will determine the role of Pumilio-mediated regulation in overall Snap25 levels as well as in Snap25 localization and local translation at the presynaptic site. Aim 2 will investigate the role of Pumilio-mediated Snap25 regulation in presynapstic terminal formation and function. I will investigate these questions using both whole-cell and compartmentalized primary rat neuronal culture and organotypic slice culture in combination with genetic manipulation of Pumilio and SNAP25. I will determine how Pumilio knockdown impacts Snap25 localization and translation, and subsequently how disruption of this regulatory process impacts presynaptic terminal formation and synaptic release. Together, the proposed experiments will provide novel insight into post- transcriptional mechanisms of Snap25 regulation during development, and how these mechanisms may play a role in synaptogenesis, synapse function, and associated neurodevelopmental disorders.